A number of antibodies, most notably Rituximab (MabThera®; Hoffmann-LaRoche, Ltd; Basel, Switzerland) and Herceptin® (Genentech, Inc.; South San Francisco, Calif.), have shown significant therapeutic value as highly selective and effective anti-tumor agents. Although these antibodies can bind to specific antigens on the tumor cells, their anti-tumor activity depends at least in part on the subsequent binding of natural killer (NK; a table of abbreviations is provided in Table Z, located after the Examples) cells to the Fc (constant) portion of the antibody with consequent destruction of the tumor cell via an antibody-dependent cellular cytotoxicity (ADCC) mechanism.
NK cells are a class of lymphocytes that typically comprise approximately 10% of the lymphocytes in a human. The primary function of NK cells is to provide an innate cellular immune response against tumor and infected (target) cells. Roles in the priming and regulation of humoral immune response, fetal development and the elimination of stressed or damaged normal cells have also been demonstrated and/or are considered to be likely. NK cells, which are characterized as having a CD3−/CD56+ phenotype, display a variety of activating and inhibitory cell surface receptors. The binding or ligation of an activating NK cell receptor to the corresponding ligand on a target cell triggers the NK cell to exert a cytotoxic effect directly against the target cell and to secrete a variety of cytokines that perform functions such as the stimulation and recruitment of other elements of the immune system to act against the target. Activated NK cells lyse target cells via the secretion of the enzymes perforin and granzyme, stimulation of apoptosis-initiating receptors and other less well characterized mechanisms.
NK cell inhibitory receptors predominantly engage with major histocompatibility complex class I (“MHC-I”) proteins on the surface of a normal cell. When so engaged, these inhibitory receptors prevent NK cells from becoming activated. The MHC-I molecules define cells as “belonging” to a particular individual. As expression of these MHC-I molecules can prevent NK cell activation toward a target cell, it is thought that NK cells can be activated only by cells on which these “self” MHC-I molecules are missing or defective, such as is often the case for tumor or virus-infected cells. The NK cell phenotype and activation pattern are distinct from that exhibited by cytotoxic T-lymphocytes (“CTLs,” CD3+/CD56−/CD8+ phenotype) that are activated by target cells that display small foreign peptide fragments derived from viruses or tumor cells attached to the surface MHC-I molecules. Scientists have speculated that NK cells evolved as a response to tumor and infected cells that evade destruction by CTLs through suppression or disruption of the display of peptide-presenting MHC-I molecules.
NK cells have been evaluated as a therapeutic agent in the treatment of certain cancers. The NK cells used for this purpose are isolated from the peripheral blood lymphocyte (“PBL”) fraction of blood from the subject, expanded in cell culture in order to obtain sufficient numbers of cells, and then re-infused into the subject. Although the results of this therapy have been promising, preparation of the autologous NK cells is expensive, labor intensive and time consuming Furthermore, quality control of these cells is complicated by each preparation being subject specific. In particular, the quantity of NK cells that can be isolated from a subject can vary substantially, and these cells are often deficient in proliferative ability and/or cytotoxic activity. Another limitation on the use of NK cells as a therapeutic agent results from the presence of surface antigens on the cells that can evoke an immune rejection response when the cells are infused into a subject other than the one from which they were isolated. This necessitates careful MHC-I cross-matching between the donor and the recipient as well as the need to immuno-suppress the recipient.
The NK-like cell line NK-92 was discovered in the blood of a subject suffering from a non-Hodgkins lymphoma. NK-92 cells lack the major inhibitory receptors that are displayed by normal NK cells, but retain the majority of the activating receptors. Characterization of the NK-92 cell line (Gong et al., 1994; Yan et al., 1998) revealed that NK-92 cells are cytotoxic to a significantly broader spectrum of tumor and infected cell types than are NK cells, and further that they often exhibit higher levels of cytotoxicity toward these targets. NK-92 cells do not, however, attack normal cells nor do they elicit an immune rejection response. In addition, NK-92 cells can be readily and stably grown and maintained in continuous cell culture and, thus, can be prepared in large quantities under c-GMP compliant quality control. This combination of characteristics has resulted in NK-92 being entered into presently on-going clinical trials for the treatment of multiple types of cancers.
Although NK-92 cells retain almost all of the activating receptors and cytolytic pathways associated with NK cells, they do not express the CD16 receptor and, therefore, cannot lyse target cells via the ADCC mechanism. This means that despite their other benefits, NK-92 cells cannot potentiate the anti-tumor and anti-infection effects of endogenous or exogenous antibodies in the manner of NK cells. Other NK-like cell lines in addition to NK-92 are also known. Some of these other NK-like cell lines express CD16, but this expression is unstable; the cells are typically difficult to grow in cell culture; and few exhibit robust cytotoxic activity. For these reasons, only NK-92 of the currently known NK-like cell lines is a viable candidate as a therapeutic agent even though it lacks CD16 and, consequently, the ability to kill target cells via the ADCC mechanism.
Thus, it would be an advantage to restore CD16 expression and the ability to act via the ADCC mechanism to NK-92 cells, thus permitting those cells to be used in concert with antibodies for therapeutic and related purposes. However, NK cell lines have been found to be recalcitrant to gene transfer, a feature that has hampered the development of such cell lines for research or therapeutic purposes. For NK-92 cells, transformation efficiencies of only 5-15% and 10-20% have been achieved using particle-mediated gene transfer or retroviral transduction (Nagashima et al., 1998; Tam et al., 1999). NK-92 cell lines that stably expresses the CD16 cell surface receptor are currently unavailable.